Functionalization of magnetic nanoparticles by creatine as a novel and efficient catalyst for the green synthesis of 2-amino-4H-chromene derivatives

By employing the naturally-originated molecule of creatine, Fe3O4@SiO2-creatine as an environmentally benign magnetic organometallic nanobiocatalyst was successfully prepared via a convenient and green route. Then to acquire an inclusive comprehension of different properties of the catalyst, it was studied by various characterization techniques such as FT‐IR, FE-SEM, TEM, EDX, XRD, and VSM analyses. It was found that the size distribution of nanoparticles was an average diameter size of 70 nm. To examine the catalytic activity, it was applied in sequential knoevenagel condensation-Michael addition room temperature reaction of dimedone, malononitrile, and different substituted aromatic aldehydes to produce a variety of 2-amino-tetrahydro-4H-chromene-3-carbonitrile derivatives in a single step. Among the multiple outstanding advantages that can be mentioned for this work, some of the most noticeable ones include: affording the products in short reaction times with high yields, operating the reaction at ambient conditions and ease of catalyst separation.

In the last decades, due to growing concerns about environmental contamination and its extensive detrimental effect on earth's ecosystems, designing more environmentally benign chemical processes has attracted substantial attention. Global interest has recently shifted into developing more eco-friendly synthetic approaches to accomplish the most important goal of green chemistry which is stopping or decreasing the use and generation of hazardous and toxic chemicals [1][2][3] . In this regard, great efforts have been made to the use of bio-based feed stocks such as cellulose 4,5 , chitosan 6,7 , alginic acid 8,9 and newly, creatine as natural supports in designing and producing different catalysts 10 . Creatine is a naturally occurring nitrogen-containing organic acid that exists primarily in muscle tissue of vertebrates and plays a very significant role in the energy supply process for muscle tissue by participating in recycling adenosine triphosphate (ATP) via giving a phosphate group to adenosine diphosphate (ADP) 10 . Creatine's natural production in the human body mainly takes place in the liver and kidneys and includes the enzyme-promoted reaction between glycine and arginine amino acids to form guanidinoacetate 11 . Then, in the second step, enzyme-assisted methylation of guanidinoacetate using s-adenosyl methionine as the methyl-group donor produce creatine. After that, the as-synthesized creatine is being transferred to the muscles through the bloodstream. Apart from the major bio-synthesized supply of creatine, it can also be obtained from the diet 12 . Since creatine is naturally originated, it doesn't have any harmful effect on living systems and affords the demand of chemists to employ green and natural materials. Having both carboxylic and amine groups, creatine is capable of activating raw materials both in acid-catalyzed reactions as well as base-catalyzed reactions; so, it has attracted chemist's attention as a potential bifunctional catalyst. Herein, by supporting this biocompatible, non-toxic and biodegradable molecule on a magnetic core, a novel reusable organometallic superparamagnetic creatine-based nanobiocatalyst was designed, prepared, characterized and then it was employed in a one-pot three-component condensation reaction for the synthesis of 2-amino-tetrahydro-4H-chromene-3-carbonitrile. In addition to have a broad spectrum of biological and pharmacological properties such as antitumor 13,14 , antibacterial [15][16][17] , antiviral 18 , antifungal 19 , anti-influenza 20 , anti-inflammatory 21 , anticancer 22,23 , and antiallergenic activity 24 45 . Typically, 4.70 g FeCl 3 ·6H 2 O and 1.72 g FeCl 2 ·4H 2 O (with a molar ratio of 2:1) were mixed with 80 mL of distilled H 2 O in a round bottom flask under N 2 atmosphere. The temperature was gradually increased up to 80 °C and at this point, 10 mL NH 3 ·H 2 O (25% v/v) was added dropwise to the vigorously stirring above-mentioned mixture. After the addition of ammonia, stirring of the mixture was continued for 45 min and then it was cooled to room temperature. The resulting black sediment was aggregate with an external magnet and was frequently washed with distilled water, ethanol and acetone in turn and dried at 70 °C in an oven in order to make it ready for further modifications.

Synthesis of Fe 3 O 4 @SiO 2 NPs.
Coating the magnetic Fe 3 O 4 core with a layer of SiO 2 was performed through a modified Stöber method 46 . Initially, 2.00 g of as-prepared Fe 3 O 4 NPs were dispersed in a mixture of 160 mL ethanol and 40 mL distilled water for 15 min using an ultrasonic water bath. Later, 10 mL of ammonia solution (25 wt%) was injected dropwise to the reaction mixture under intensive magnetic stirring. In the next step, 1 mL of TEOS was dropped slowly into the solution by a syringe for 20 min. in the last step, the mixture allowed to stir for 12 h at room temperature. Finally, the brown obtained solid was collected by a magnet and after successive washing with distilled water and ethanol several times it was dried at 60 °C.
Preparation of chloropropyl-functionalized Fe 3 O 4 @SiO 2 MNPs. Binding (3-chloropropyl)-trimethoxysilan (CPTMS) as a linker to silica-coated MNPs was performed following the procedure of one of previous works 47 . Firstly, 1 mL of CPTMS reagent was dissolved in 100 mL of dried toluene. afterward, 1.00 g of Fe 3 O 4 @SiO 2 was added to this mixture and the solution stirring last for 18 h at 60 °C. The resulted brown precipitate (Fe 3 O 4 @ SiO 2 -Cl) was washed with toluene, separated by a permanent magnet, and vacuum dried at 70 °C.

Synthesis of Fe 3 O 4 @SiO 2 -creatine.
At first, 1.00 g of as-prepared Fe 3 O 4 @SiO 2 -Cl nanoparticle and 1.31 g (10 mmol) of creatine were added to 80 mL of ethanol and the mixture was stirred in a round bottom flask under refluxing conditions for 10 h. Then, the obtained solid substance was magnetically separated, washed repetitively with distilled water, ethanol and acetone and dried at 70 °C in an oven.
General procedure for the synthesis of 4H-chromene derivatives (4a-o). First, 1.00 mmol (0.14 g) dimedone, 1.00 mmol of aromatic aldehydes and 1.10 mmol (0.07 g) malononitrile were mixed together in 2 mL of ethanol in presence of 0.04 g of the prepared catalyst. This mixture stirred for appropriate times at room temperature. The progress of the reaction was monitored by TLC (ethyl acetate: n-hexane, 1:1). After completion of the reaction, the catalyst was separated using an external magnet and the obtained precipitates which were the products, recrystallized in ethanol to gain highly pure crystalline 4H-chromene compounds.

Results and discussion
In this study, a unique bifunctional nanobiocatalyst with acidic and basic sites was designed by immobilizing creatine on the surface of Fe 3 O 4 @SiO 2 NPs. At first, Fe 3 O 4 @SiO 2 NPs were synthesized. In the second step, these NPs were modified by biomolecule of creatine as an environmentally friendly and low-cost compound ( Fig. 1).
Several identification techniques such as FT-IR, EDX, FE-SEM, TEM, and VSM were employed to study the properties of the nanocatalyst. Also, after several times using it as a bifunctional catalyst for the synthesizing of 2-amino-4H-chromenes, there was no specific changing in the structure and activity of the mentioned catalyst.  44 . Additionally, peaks appeared at about 3300 cm −1 are assigned to the asymmetric stretching vibration of hydroxyl groups on the surface of the silica layer. Coating of magnetic core with the organo-layer of creatine is confirmed by stretching vibrations appeared at 1683 cm −1 which is related to C=O functional group of creatine biomolecule (Fig. 2b) 51 . Peaks appeared in the IR spectrum of the composite are very good in agreement with functional groups existing in the structure of the organo-functionalized MNPs and could be counted as evidence to approve the core-shell structure of as-described nanocatalyst.
EDX analysis. EDX analysis was performed to determine the elements in the composition of the catalyst. The result of EDX analysis is demonstrated in Fig. 2c. It clearly indicated the existence of iron, silicon, carbon, oxygen and nitrogen elements in the nanobiocomposite. The presence of the Fe peaks in the EDX result indicates the existence of Fe 3 O 4 nanoparticles in the structure. In addition, silicon and nitrogen peaks are clear signs of TEOS and creatine, respectively. Carbon and oxygen peaks are related to the presence of these elements in TEOS and creatine molecules. The observed oxygen peak is also related to Fe 3 O 4 nanoparticles.

FE-SEM image study.
In order to identify the surface morphology and particle size distribution of the nanocatalyst FE-SEM imaging technique was used ( Fig. 3a-d). As can be observed in SEM micrographs in four different magnifications, the Fe 3 O 4 @SiO 2 -creatine nanoparticles have an approximately spherical shape and size distribution with an average diameter length of 70 nm. To determine the structure of interior layers of the synthesized nanobiocatalyst and to prove the core-shell structure of it, TEM analysis was utilized. As it is observable in Fig. 4. TEM images were taken in 3 different magnifications 150 nm (Fig. 4a), 60 nm (Fig. 4b), 30 nm (Fig. 4c) which clearly show the shell consisting of silica inorganic layers and organic layer of creatine that have coated Fe 3 O 4 core.
VSM analysis. Magnetic characteristic of the Fe 3 O 4 @SiO 2 -creatine nanocatalyst was measured via VSM analysis at room temperature (300 K). Figure 5 shows the magnetization as a function of magnetic field strength. The saturation magnetization value (Ms) of uncoated MNPs (Fe 3 O 4 ) (Fig. 5a) was found to be 56.0 emu g −152 . With sweeping the magnetic field from − 20,000 to + 20,000 oersted, the saturation magnetization value (Ms) of the prepared nanocomposite was found to be about 22.5 emu g −1 (Fig. 5b), which is lower than neat Fe 3 O 4 NPs. This reduction in the saturation magnetization is mostly ascribed to the existence of shells that have wrapped the Fe 3 O 4 core. Despite this reduction in the saturation magnetization of catalyst, the intensity of its magnetic power is still high and can be easily separated from the reaction media by an external magnet. Moreover, the resultant hysteresis loop clearly certifies the superparamagnetic characteristic of the prepared nanocomposite.   Application of the nanobiocatalyst in organic synthesis. In order to investigate the catalytic activity of the created nanocatalyst, it was utilized in the one-pot synthesis of 2-amino-7,7-dimethyl-5-oxo-4-arylhexahydro-4H-chromene-3-carbonitrile derivatives. To optimize the reaction condition, the reaction between dimedone 3, 4-isopropylbezaldehyde 2, and malononitrile 1 (with a mole ratio of 1:1:1.1) in 2 mL of ethanol as a green solvent and at room temperature, was chosen as the model reaction. www.nature.com/scientificreports/ At first, the effect of catalyst amount on the reaction rate and yield was studied. Using 0.04 g of the nanocatalyst was found to be adequate to catalyze the reaction and produce high yields of 4H-chromene derivatives. According to Table 1, in the presence of lower amounts of catalysts, lower yields of the products were obtained and the reaction took a longer time to complete. With increasing the amount of nanocatalyst no significant improvement in the reaction rate and yield was observed. As shown in Table 1, only trace amounts of the desired products were obtained in the absence of the nanocatalyst. To optimize the solvent of the reaction, the effect of H 2 O as another green solvent has also been studied. Finally, based on the results, ethanol was found to be the most suitable solvent (Table 1, entry 7). Since the desired products were produced in high to excellent yield at room temperature, testing higher temperatures were not needed.
In order to compare the efficiency of the prepared catalyst with its substrate before coating with creatine, the model reaction was performed with 0.04 g of each of the catalysts mentioned in Table 2. As can be seen in the table, at a specified time the efficiency of the prepared catalyst is much more than the substrates alone. Fe 3 O 4 allows the catalyst to be easily separated from the reaction medium by an external magnet. In addition, Fe 3 O 4 as Lewis acid can increase the reaction rate. Creatine as a proton donor agent activates electrophiles and also  In order to show the repeatability of this approach and to generalize the optimum conditions to other aromatic aldehyde a wide range of substituted benzaldehydes bearing both electron-withdrawing and electron-donating groups were chosen and the reactions of those aldehydes (2) with malononitrile (1) and dimedone (3) has led to the formation of a diverse sort of 2-amino-4H-chromene-3-carbonitrile derivatives under optimum reaction conditions. The results presented in Table 3 show all products were successfully synthesized in good-to-excellent yields after suitable reaction time. Moreover, the results indicated that aldehydes possessing electron-withdrawing groups reacted much faster and gave higher yields than those bearing electron-releasing groups, however, the yield of the products with aldehyde bearing electron-releasing groups was completely satisfying in comparison with previous works [53][54][55] .
The suggested possible mechanism of the reaction in the presence of the nanocatalyst is described in Fig. 7. Creatine as a bifunctional molecule can significantly catalyze the chromene formation reaction. Initially, the acidic hydrogen atom of the catalyst activates the aromatic aldehyde through protonation of its carbonyl group. So, the electrophilicity of the carbonyl group increases. Concurrently, the Nitrogen atoms in the guanidine part  Table 1. Optimizing the reaction conditions in the synthesis of 2-amino-4H-chromene derivatives. a The yields refer to the isolated product 4h.    In addition, the catalytic activity of Fe 3 O 4 @ SiO 2 -creatine was evaluated in comparison with other catalysts, and the results are shown in Table 4. As can be seen, the Fe 3 O 4 @SiO 2 -creatine catalyst performs better than other studies.

Entry Catalyst loading (g) Solvent Temp (°C) Time (min) Yield a (%)
Catalyst recyclability. The reusability of the catalyst was assessed in the synthesis of 4H-chromene derivatives under optimum reaction conditions. Owing to its magnetic nature, the catalyst was easily separated from the reaction mixture by an external magnet, washed repeatedly with ethanol and distilled water, dried and reused after each run. It was observed that the catalyst could be reused for at least six times without any significant loss in its activity (Fig. 8). The result of EDX analysis (Fig. S19) and FT-IR spectrum (Fig. S20) of the reused catalyst showed no change in the composition of the catalyst.

Conclusions
In summary, a novel and green renewable creatine-functionalized magnetic nanobiocomposite catalyst was designed and prepared via a facile, simple and cost-effective method beginning from readily accessible and nontoxic starting materials. Subsequently, in order to investigate the catalytic activity of the prepared nanocomposite, it was employed in the one-pot 3-CR synthesis of biologically-active 2-amino-7,7-dimethyl-5-oxo-4-aryl-hexahydro-4H-chromene-3-carbonitrile derivatives. The products were obtained with the yields of 81-96% within a few minutes. Multiple characterization techniques such as FT-IR, EDX, FE-SEM, XRD, TEM, and VSM were used to analyze various properties of the as-synthesized organocatalyst. The FE-SEM images showed that the average size of NPs was about 70 nm. The result of other applied identification techniques, all confirmed the formation of the core-shell spherical structure of the nanocatalyst. The advantages of this efficient environmentally-friendly protocol include ease of preparation and separation, providing safe and green reaction conditions, high atom economy and excellent yields in relatively short reaction times, magnetically recyclability of the heterogeneous catalyst, clean, simple and easy work-up procedure.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.